1

Single dose pharmacodynamics of amphotericin B against Aspergillus species in an in vitro 1

pharmacokinetic/pharmacodynamic model 2

3

Running title: Single-dose AMB pharmacodynamics for Aspergillus spp 4

5

6

Rafal Al-Saigh1, Maria Siopi1, Nikolaos Siafakas1, Aristea Velegraki2, 7

Loukia Zerva1, Joseph Meletiadis1 8

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Clinical Microbiology Laboratory, Attikon Hospital1, Mycology Research Lab, Department of 11

Microbiology2, Medical School, National and Kapodistrian University of Athens, Athens, Geece 12

13

14

15

16

Correspondence: Joseph Meletiadis, Ph. D. 17

Lecturer in Mycology, 18

Clinical Microbiology Laboratory, 19

Attikon University Hospital 20

Rimini 1, Haidari, 124 62 Athens 21

Tel: 210-583-1909 22

Fax: 210-532-6421 23

Email: jmeletiadis@med.uoa.gr 24

Copyright © 2013, American Society for Microbiology. All Rights Reserved.Antimicrob. Agents Chemother. doi:10.1128/AAC.02484-12 AAC Accepts, published online ahead of print on 28 May 2013

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ABSTRACT 25

Conventional MIC testing of amphotericin B results in narrow MIC ranges challenging the 26

detection of resistant strains. In order to discern amphotericin B pharmacodynamics, the in vitro 27

activity of amphotericin B was studied against Aspergillus isolates with the same MIC with a new 28

in vitro pharmacokinetic/pharmacodynamic (PK/PD) model that simulates amphotericin B human 29

plasma levels. Clinical isolates of A. fumigatus, A. terreus and A flavus with the same CLSI modal 30

MICs of 1 mg/l were exposed to amphotericin B concentrations following the plasma 31

concentration-time profile after single bolus administration with Cmax 0.6, 1.2, 2.4 and 4.8 mg/L. 32

Fungal growth was monitored up to 72h based on galactomannan production. Complete growth 33

inhibition was observed only against A. fumigatus with amphotericin B Cmax ≥2.4 mg/L. At lower 34

Cmaxs 0.6 and 1.2 mg/L, a significant growth delay of 34h and 52h was observed, respectively 35

(p<0.001). For A. flavus, there was no complete inhibition but a progressive growth delay of 1h-50h 36

at amphotericin B Cmax 0.6-4.8 mg/L (p<0.001). For A. terreus, the growth delay was modest (up to 37

8h) at all Cmaxs (p<0.05). The Cmax (95% confidence interval) associated with 50% activity for A. 38

fumigatus was 0.60 (0.49-0.72) mg/L, significantly lower than for A. flavus 3.06 (2.46-3.80) and for 39

A. terreus 7.90 (5.20-12.29) (p<0.001). A differential in vitro activity of amphotericin B was found 40

among Aspergillus species despite the same MIC in the order of A. fumigatus>A flavus>A. terreus 41

in the in vitro PK/PD model possibly reflecting the different concentration- and time-dependent 42

inhibitory/killing activities amphotericin B exerting against these species. 43

44

45

Keywords: amphotericin B, A. fumigatus, A. flavus, A. terrus, pharmacodynamics, 46

pharmacokinetics, simulation, in vitro 47

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INTRODUCTION 49

Amphotericin B (AMB) is an antifungal drug of major importance in the treatment of 50

invasive aspergillosis (1). It is a highly lipophilic and amphoteric molecule that interacts with 51

fungal cell membrane forming pores and disrupting its integrity (2). Due to its unique mechanism of 52

action, it demonstrates a wide range of pharmacodynamic effects and broad spectrum of antifungal 53

activity. However, conventional MIC testing of amphotericin B resulted in narrow MIC ranges 54

within 1-2 twofold dilutions challenging the detection of resistant strains (3-5). Efforts to develop in 55

vitro assays that separate susceptible and resistant strains using richer media or gradient drug 56

concentrations strips were unsuccessful (3, 5). Species-specific epidemiological cutoff values 57

(ECV) have been proposed for amphotericin B and Aspergillus spp. based on CLSI broth 58

microdilution methodology with A. terreus ECV being one dilution higher than A. fumigatus and A. 59

flavus ECV (6). 60

In addition to inhibitory activity captured by the MIC, amphotericin B exerts a range of 61

different pharmacodynamic effects such as post-antifungal effect and concentration-dependent 62

killing (7). All these effects are usually determined after fungal exposure to constant drug 63

concentrations (2). However, in vivo, fungus is exposed to non-constant amphotericin B 64

concentrations as the drug undergoes metabolism, distribution and excretion. In particular, its 65

plasma levels follow a triphasic time-concentration profile characterized by the alpha-phase 66

observed within the first 4h after administration with a half-life of <1h, the beta-phase observed 67

within 4-24h after administration with a half life of 6-10h, and the gamma phase observed >24h of 68

administration with a half-life of >120h (8). Simulating this time-concentration profile in vitro is a 69

challenge because amphotericin B binds to plastic surfaces and degrades over time (9). 70

We recently developed an in vitro model that simulated human pharmacokinetics of 71

antifungal drugs and enabled to study the pharmacodynamics of decreasing drug concentrations as 72

in human plasma (10). This pharmacokinetic/pharmacodynamic (PK/PD) model showed 73

considerable differences of voriconazole activity against Asperillus species which had the same 74

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MICs indicating that studying the in vitro activity of decreasing drug concentrations provides 75

unique information of pharmacodynamic effects of antifungal drugs (11). With this model, the time- 76

and concentration-dependent pharmacodynamic properties of antifungal drugs can be studied and 77

PK/PD analysis simulating human pharmacokinetics can be performed. 78

We therefore studied the activity of amphotericin B against A. fumigatus, A. flavus and A. 79

terreus strains with similar MICs with the new in vitro PK/PD model simulating single-dose 80

pharmacokinetics of amphotericin B in human plasma and monitoring Aspergillus growth over time 81

with galactomannan production. Despite the same MICs, important pharmacodynamic differences 82

were found among the three species with amphotericin B being less active against A. flavus and A. 83

terreus than against A. fumigatus reflecting differences in inhibitory, killing and post-drug exposure 84

effects. 85

86

MATERIAL AND METHODS 87

Strains. Three clinical strains of A. fumigatus, A. flavus and A. terreus isolated from patients 88

with invasive pulmonary aspergillosis were studied. The minimal inhibitory concentrations (MIC) 89

as determined thrice with the CLSI broth microdilution method were 1-1 mg/L for A. fumigatus, 1-1 90

mg/L for A. flavus and 1-2 mg/L (mode 1 mg/L) for A. terreus (12, 13). The A. terreus strain was 91

included because of its known reduced susceptibility to amphotericin B. The strains were 92

maintained at-70oC in 10% glycerol and cultured twice in Sabouraud Dextrose agar at 30oC for 5-7 93

days. A conidial suspension was prepared in normal saline with 1% Tween 20. Conidia were 94

counted with a Newbauer chamber in order to obtain a final suspension 1x105CFU/ml and their 95

concentration was confirmed by quantitative cultures on Sabouraud Dextrose Agar. 96

Antifungal susceptibility testing. In order to explore the in vitro susceptibility of the three 97

isolates with other methodologies, the isolates were also tested with the gradient concentration strip 98

method Liofilchem™ MIC Test Strips (MTS) (Varelas SA, Athens, Greece) according to 99

manufacturer’s instructions and the XTT methodology as previously described (14). Briefly, for 100

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MTS method agar plates with RPMI1640+MOPS+2% glucose were inoculated in three directions 101

with a cotton swab dipped into a 0.5MacFarland conidial inoculum and the MTS was applied and 102

incubated at 35oC for 24h and 48h. The MIC was determined as drug concentration at which the 103

border of the elliptical inhibition zone corresponding to 100% inhibition intersected the strip. For 104

XTT methodology, twofold serial dilutions of amphotericin B in RPMI1640+MOPS in 96-105

flatbottom well microtitration plates were inoculated with 1-5x104CFU/ml, incubated for 48h when 106

0.1 mg/ml XTT + 25 μM Menadione was added in each well, further incubated for 2h at 35οC when 107

absorbance at 450nm was measured and % growth in each well was calculated in comparison to 108

growth in the drug-free control. The MIC was determined as the lowest drug concentration with 109

<10% growth. Furthermore, the minimal fungicidal concentration (MFC) was determined with an 110

XTT methodology as previously described (13). Briefly, after XTT MIC determination, fresh 111

medium was added to all clear wells after washed with saline and after incubation for 24h at 35oC 112

XTT+MEN was added and % growth was calculated based on absorbance at 450nm. The MFC was 113

determined as the lowest drug concentration showing <10% growth. All tests were performed three 114

times. 115

Antifungal drug and medium. Amphotericin B (AMB, Fungizone, Bristol-Myers) was 116

reconstituted at 10,000 mg/L according to manufacturer’s instructions and stored at-70oC. The 117

medium contained 10.4 g/L RPMI1640 with glutamine without sodium bicarbonate (Sigma-118

Aldrich, St. Luis, MO) and 0.165M buffer MOPS (Invitrogen, Carlsbad, CA), pH 7.0, with 100 119

mg/L chloramphenicol (Sigma -Aldrich, St. Luis, MO). 120

In vitro pharmacokinetic/pharmacodynamic model. The in vitro pharmacokinetic 121

simulation model consists of a) a glass beaker containing 700ml medium (external compartment 122

EC) in which is placed, b) a dialysis tube of 10 ml volume (internal compartment IC) the wall of 123

which consists of cellulose permeable membrane allowing the free diffusion of molecules with a 124

molecular weight <20kD, and c) a peristaltic pump (Minipuls Evolution, Gilson, France), which 125

removes the content of EC and adds medium within it at a rate equivalent to drug removal from 126

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human serum (10). The conidial suspension is inoculated in the IC within which the growing fungus 127

and its derivative galactomannan (molecular weight 20-60kD) remain trapped, while nutrients and 128

drug diffuse freely between IC and EC. The concentration of the galactomannan increases with 129

fungal growth. The drug is injected into the EC and its concentration is adjusted by the pump to 130

correspond to the average half-life observed in human plasma after intravenous administration of 131

amphotericin B. The EC was covered with aluminum foil in order to minimize light exposure and 132

placed on a heated magnetic stirrer (37oC). Before starting each experiment, temperature and flow 133

rate were controlled. All experiments were repeated twice. 134

Determination of amphotericin B concentrations. The drug levels in the IC were 135

determined by a microbiological method using the strain Paecilomyces variotii ATTC 22319, 136

susceptible to AMB (15). Specifically, P.variotii conidia at final concentration 5x105CFU/ml were 137

inoculated into prewarmed at 54oC RPMI1640 medium + MOPS with 15 g/L agar and poured to 138

plastic plates 10x10cm. After solidification of the agar, 1 cm-diameter holes were opened and filled 139

with 100μl of known drug dilutions (range 0.25-16 mg/L), as well as 100μl of IC samples. The 140

plates were incubated at 37oC for 24h when diameters of inhibition zones were measured. Unknown 141

drug concentrations in the IC samples were determined using the standard curve constructed from 142

known drug concentrations and corresponding diameters of inhibition zones. 143

Pharmacokinetic analysis. Several clinically relevant AMB doses (0.25, 0.5, 1 and 1.5 144

mg/kg) were simulated in the in vitro model with maximum concentrations in human plasma Cmax 145

of 0.6, 1.2, 2.4 and 4.8 mg/L and AUC values of 9.4, 21, 46.3 and 57.3 (8, 16, 17). After taking 146

into account any loss of amphotericin B during the experiments due to degradation and surface 147

binding, the flow rate was adjusted in order to approximate the plasma concentration profile of 148

amphotericin B in humans with an alpha phase with a short half life of <1h observed within 4h after 149

drug administration followed by a beta phase with a longer half life of 6-10h observed 4-24h after 150

drug administration and a gamma phase with a half life of 120h observed >24h after drug 151

administration (8). Amphotericin B concentrations were determined at 0h, 4h, 6h, 8h, 20h, 24h, 152

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44h, 48h and 72h after the introduction of the drug in the IC using the bioassay. The data were 153

analyzed by nonlinear regression based on a three-compartment model described by the equation 154

C= Cαekαt+Cβekβt+Cγe

kγt where kα, kβ and kγ are the rate constants, Cα, Cβ and Cγ are the Y-155

intercepts for alpha, beta and gamma phase, respectively, and C is the concentration at a given time 156

t. The half-lives of alpha, beta and gamma phases were calculated for EC and IC separately using 157

the equations t1/2.α=kα/ln(2), t1/2.β=kβ/ln(2), and t1/2.γ=kγ/ln(2), respectively, and were compared with 158

the corresponding values observed in human plasma. 159

Determination of fungal growth. Fungal growth in the IC was assessed in samples of 160

100μl at regular time intervals by determining galactomannan production using an ELISA (Platellia, 161

Biorad, Athens, Greece). Samples were diluted with 200μl saline in order to reach the final volume 162

of 300μl before processing. Results were expressed as a galactomannan index (GI) according to the 163

manufacturer's instructions. Galactomannan levels were also determined in the EC in order to 164

ensure that no galactomannan was escaped from the IC. 165

Real time PCR conidial equivalent was used as an alternative biomarker of fungal growth 166

and killing. Aspergillus DNA was extracted from 200μl samples from the IC of the in viro PKPD 167

model after 0h and 72h with the Qiagen DNA Blood Mini kit (Roche Diagnostics, Athens, Greece) 168

after enzymatic (incubation with protenase K at 56oC for 10 min) and mechanical (1 min vortex 169

with glass beads) extraction as previously described (18). Real time PCR was performed with a 170

previously described assay (2Asp assay) using Aspergillus specific primers (ASF1 and ADR1) and 171

probe (ASP28P) (19). The threshold cycle (Ct) of each sample, which identifies the cycle number 172

during PCR when fluorescence exceeds a threshold value determined by the software, was 173

converted to conidial equivalent (CE) A. fumigatus DNA by interpolation from a 6-point standard 174

curve of Ct values obtained with 103-108 Aspergillus CFU/ml. The reduction of the PCR CE after 175

72h of incubation compared to 0h was calculated for each species and amphotericin B doses. 176

Pharmacodynamic analysis. In vitro pharmacodynamics of each amphotericin B dose and 177

Aspergillus species were determined based on the GI-time relationship analyzed with the Emax 178

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model: E = Emin + Emax * Tγ / (Tγ + T50), where E is the GI (dependent variable), Emax and Emin , the 179

maximum and minimum GI, respectively, T the incubation time (independent variable), T50 the 180

time corresponding to 50% of Emax and γ the slope of the curve. In addition, the area under the 181

galactomannan index-time curve (AUCGI) was calculated for each amphotericin B dose. As shown 182

previously, the parameters Emax, γ and T50 describe the extent, rate and time of galactomannan 183

production, respectively, whereas the AUCGI is a surrogate marker of fungal growth. The higher the 184

AUCGI, the greater is the fungal growth. The percentage of fungal growth at each dose was 185

calculated based on the AUCGI of each doses divided by the AUCGI of the growth control. Based on 186

all these parameters, the in vitro activity of amphotericin B dose against each Aspergillus species 187

was estimated. Finally, the in vitro PKPD relationship AUCGI-Cmax was plotted for each species and 188

analyzed with the Emax model. 189

Statistical analysis. All analysis was performed with the software Prism 5.01 (GraphPad 190

Inc., La Jolla, CA). All Emax models were globally fitted to the data with Emax and Emin shared 191

among data sets. Comparisons between Emax model parameters of different amphotericin B doses 192

and Aspergillus species were assessed using extra sum-of-squares F test. A p value <0.05 was 193

considered statistically significant. 194

195

RESULTS 196

Antifungal susceptibility testing. The MTS MICs for A. fumigatus, A. flavus and A. terreus 197

were 0.75-0.75, 2-3 and 1-1.5 mg/l after 24h and 2-3, >32, and >32 mg/l after 48h, respectively. 198

The XTT MICs and MFCS were 1-2 and 1-2 mg/l for A. fumigatus, 2-2 and 2-4 mg/l for A. flavus 199

and 1-2 and 8-16 mg/l for A. terreus. 200

Bioassay for amphotericin B. The amphotericin B concentrations detected with the 201

bioassay ranged from 0.25 mg/L to 16 mg/L and lowest limit of detection was 0.25 mg/L. Across 202

all experiments performed on the same and different days, the diameter of inhibition zone correlated 203

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linearly with amphotericin B concentration (r2>0.77, global r2=0.84). The coefficient of variation 204

ranged from 5% to 25% (average 15%) for all concentrations. 205

Pharmacokinetic analysis. The Cmax in the IC were 0.76-0.78, 1.05-1.10, 2.5-2.7 and 3.9-206

4.4 mg/L and the AUCs 4.5-5, 8-8.6, 31.9-33.2, 64.8-67.9 mg.h/l, respectively, with t1/2,α 0.2-2h, 207

t1/2,β 10-17h and t1/2,γ 71h for the simulated amphotericin B doses 0.25, 0.5, 1 and 1.5 mg/kg, 208

respectively. Because of the low detection limit of the bioassay, a gamma phase was observed only 209

for the highest dose of amphotericin B with Cmax 4.8 mg/L. These values were similar to those 210

observed in human plasma after administration of amphotericin B doses 0.25-1.5 mg/kg with the 211

largest deviations observed at lower doses (Figure 1). 212

Pharmacodynamic analysis (PD). The GI–time curves were described very well with the 213

Emax model (R2> 0.86) and they were characterized by the same Emax but different slopes and T50s 214

for the different amphotericin B doses and Aspergillus species. Among all species and doses tested, 215

complete inhibition of galactomannan production was observed only against A. fumigatus with 216

amphotericin B doses corresponding to Cmax ≥2.4 mg/L (Figure 2). At lower doses, a significant 217

delay in galactomannan production was observed with a mean±SEM T50 of 38.1±2.3h for Cmax 0.6 218

mg/L and 57.9±2.7h for Cmax 1.2 mg/L compared to 4.2±0.4h for the drug free control (p<0.001). 219

For A. flavus, there was no complete inhibition but a progressive delay of galactomannan 220

production with increasing amphotericin B doses since the T50 increased from 8.2±0.6h for the 221

growth control to 9.3±0.6h at amphotericin B dose with Cmax 0.6 mg/L, 24.3±3.2h at Cmax 1.2 mg/L, 222

36.7±3.1h at Cmax 2.4 mg/L and 57.8±2.7h at Cmax 4.8 mg/L (p<0.001). For A. terreus, the delay in 223

galactomannan production was modest since the T50 of 4±0.4h for the growth control increased to 224

12.6±3.3h at the highest dose of amphotericin B with Cmax 4.8 mg/L (p=0.013). The differences 225

among the tree species in galactomannan production with the two high doses of amphotericin B 226

with Cmax 2.4 and 4.8 mg/l were confirmed with real time PCR results with PCR CE at 72h being 227

reduced by 0.7 and 0.8 log10CE of A. fumigatus, 0.1 and 0.4 log10CE of A. flavus and increased by 228

1.5 and 0.1 log10CE of A. terreus, respectively (data not shown). 229

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Finally, the in vitro activity of amphotericin B against the three Aspergillus species was 230

compared by constructing PK/PD curves. In order to quantify the effect of each amphotericin B 231

dose at the entire 72h period of incubation, the AUCGI was calculated for each dose and species as a 232

surrogate marker of fungal growth and plotted against the corresponding Cmaxs (Figure 3). The in 233

vitro PK/PD relationship followed a sigmoid pattern (global R2=0.99). The Cmax (95% confidence 234

interval) associated with 50% activity for A. fumigatus was 0.60 (0.49-0.72), which was statistically 235

significant lower than the corresponding Cmax against A. flavus (3.06, 2.46-3.80) and A. terreus 236

(7.90, 5.20-12.29) (p<0.001). 237

238

DISCUSSION 239

Important pharmacodynamic differences of amphotericin B against the three Aspergillus 240

species were found in the recently developed in vitro PK/PD model where conidia were exposed to 241

decreasing drug concentrations simulating the plasma concentration-time profile of amphotericin B. 242

Despite the same MICs, the strongest in vitro activity of amphotericin B was found against A. 243

fumigatus followed by A. flavus and A. terreus. The XTT and gradient concentration strips methods 244

showed minor differences in the MIC of the three isolates which clustered within 1-2 twofold 245

dilution, emphasizing the problem of narrow amphotericin B MIC ranges with these techniques. 246

However, 48h MTS MICs were similar for A. flavus and A. terreus and higher for A. fumigatus 247

whereas XTT MFCs were similar for A. fumigatus and A. flavus and higher for A. terreus 248

advocating for the different pharmacodynamic effects amphotericin B possessed against different 249

Aspergillus species. 250

Studying the effect of decreasing concentrations of antifungal drugs provides information 251

about pharmacodynamic properties related with sub-MIC effect, post-antifungal effect, time- and 252

concentration-dependent activities. These effects can be quantified by a surrogate marker of fungal 253

growth based on galactomannan production kinetics which captures any difference regarding the 254

above antifungal effects. Differential antifungal activity was also previously found with the present 255

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in vitro PK/PD model for voriconazole against the three Apergillus species with identical MICs 256

emphasizing the importance of studying non-constant drug concentrations (10, 11). 257

The findings of the present study are in agreement with previous time-kill assays where 258

supra-MIC (4x and 20xMIC) concentrations of amphotericin B killed A. fumigatus but not A. 259

terreus (20). Minimal fungicidal concentrations of amphotericin B were similar against A. 260

fumigatus and A. flavus and much higher against A. terreus. However, MFC/MIC ratios for A. 261

fumigatus were lower than those for A. flavus differentiating the in vitro activity of amphotericin B 262

against these two species (13). In addition, the three species were previously found to differ also in 263

the post-drug exposure effects since 4x and 1xMIC of amphotericin B demonstrated >4h post 264

antifungal effect against A. fumigatus and <4h against A. flavus and A. terreus (21). Time-265

dependent activity of amphotericin B inhibition also differed among the three Aspergillus species 266

(22). Exposure of Aspergillus conidia to supra-MIC concentrations for 8h resulted in significant 267

amount of metabolic activity for A. terreus (16%), less for A. flavus (8%) and even lesser for A. 268

fumigatus (5%) isolates. Furthermore, despite the same concentration-effect curves of amphotericin 269

B for A. fumigatus and A. flavus at 48h, the inhibitory concentration-effect curve after 8h of 270

exposure to amphotericin B were shifted to the left for A. fumigatus but not for A. flavus indicating 271

that amphotericin B activity is faster against A. fumigatus than A. flavus species (22). 272

Taking into account all these different effects exerted by amphotericin B, the order of 273

amphotericin B in vitro activity demonstrated by the present model (A. fumigatus>A. flavus>A. 274

terreus) could be explained by a fast inhibitory action and increased killing rate against A. 275

fumigatus, a slower inhibitory action and reduced killing efficiency against A. flavus and the 276

slowest inhibitory action and no killing against A. terreus as also found with real time PCR results. 277

In particular, the delayed galactomannan production of A. fumigatus but not A. flavus at Cmax=0.6 278

mg/L indicates a strong sub-MIC effect of amphotericin B against the former species. Although 279

there are no data on sub-MIC effects of amphotericin B against Aspergillus spp. such effects were 280

described against Candida spp.(23). At Cmax=1.2 mg/L, galactomannan was detected after 48h 281

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incubation for A. fumigatus reflecting the minimal fungicidal action at this concentration (usually 282

observed at 2xMIC) (13) and the long post-antifungal effect observed at 1xMC (21) together with a 283

sub-MIC effect possibly occurred after amphotericin B concentrations fell below the MIC. The 284

absence of galactomannan production at concentrations >2xMIC reflects the fungicidal activity 285

amphotericin B demonstrated at time-kill assays (20). For A. flavus, galactomannan was detected at 286

Cmax=4.8, 2.4 and 1.2 mg/L after 48h, 24h and 6h as soon as the concentration fell below MIC 287

reflecting the absence of killing and post-antifungal effects as previously described (21). Of note, at 288

all three doses galactomannan production was detected after 4h despite amphotericin B 289

concentrations being higher than the MIC reflecting the slow inhibitory action of amphotericin B 290

against this species, as previously found (22). Finally, the modest delay in galactomannan 291

production of A. terreus at all doses reflects the lack of killing, post-antifungal and possibly sub-292

MIC effect and the slow inhibitory action against this species. Thus, single-dose pharmacodynamics 293

in the present in vitro PK/PD model where amphotericin B concentrations decrease over time may 294

reflect concentration- and time-dependent inhibitory and killing activities described by MFC, time-295

kill and post-antifungal effect assays. 296

Amphotericin B was for decades the treatment of choice for aspergillosis. Clinical and 297

animal data indicated different drug efficacy against infections caused by various Aspergillus 298

species (24). Lack of in vivo efficacy, however, was not associated with significantly increased 299

MIC values (3, 25, 26), which remained similar for all three species examined in the present study 300

(13, 27). Results obtained by the new in vitro model revealed striking differences in efficacy of 301

amphotericin B against the three Aspergillus species despite their similar MICs with the following 302

order: A. fumigatus>A. flavus>A. terreus. These findings are in agreement with previous 303

comparative animal studies where treatment with amphotericin B was more effective against 304

experimental infection caused by A. fumigatus than infection with A. flavus and less effective 305

against infection with A. terreus (4, 20). In particular, amphotericin B treatment of guinea pigs 306

infected with an A. flavus or an A. fumigatus strain (each with MIC of 1 mg/L), resulted in 0% and 307

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80% survival, respectively at the highest dosage of 2.5 mg/kg (4, 20). Furthermore, the in vivo 308

PKPD parameter Cmax/MIC associated with near maximum survival in an animal model of 309

experimental aspergillosis by A. fumigatus was previously found to be 2.4 similar to the Cmax/MIC 310

ratio found in the present study to be associated with the maximum suppressive effect of 311

amphotericin B against A. fumigatus (28). However, differences in pathogenesis and virulence 312

among these species may confound in vitro-in vivo correlation (29, 30). Clinical studies also 313

demonstrated a higher mortality rate of infections by A. terreus compared to those by A. fumigatus 314

despite amphotericin B therapy (31, 32). It seems that the new in vitro model, described here, may 315

better characterize the pharmacodynamic characteristics of amphotericin B against the most 316

clinically significant Aspergillus species than conventional in vitro susceptibility systems. 317

In summary, the in vitro model simulated well amphotericin B human pharmacokinetics 318

and demonstrated a differential in vitro activity against the three Aspergillus species that was not 319

reflected by their respective MICs. The effects observed in the in vitro PK/PD model may be the 320

sum of concentration- and time-dependent inhibitory/killing activities exerted by amphotericin B 321

with the greatest activity found against A. fumigatus and the lowest against A. terreus. Future 322

studies should focus on testing larger collections of isolates in order to describe the distribution of 323

this new pharmacodynamic effect and taking into account protein binding and amphotericin B 324

disposition in human body in order to obtain clinically relevant drug exposures that was not 325

obtained with the current model. A composite pharmacodynamic effect that describes the different 326

in vitro activities of amphotericin B may overcome the MIC clustering, assess better antifungal 327

activity and help distinguish susceptible and from resistant strains. 328

329

FUNDING 330

This study was supported by the Marie Curie Reintegration Grant MIRG-CT-2007-208796 331

of the European Commission. 332

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0 6 12 18 24 30 36 42 48 54 60 66 720.0

0.5

1.0

1.5

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2.5

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In vitro pharmacokineticsof Amphotericin B

Internal compartmentExpected

Time(h)

Co

nce

ntr

atio

n (

mg

/l)

446

Figure 1. Pharmacokinetic analysis of simulated amphotericin B doses 0.3, 0.5, 1 and 1.5 mg/kg in 447

humans (dashed lines) and in the in vitro pharmacokinetic/pharmacodynamic model (solid lines) 448

with Cmax 0.6 (light gray), 1.2 (medium gray), 2.4 (dark gray) and 4.8 mg/L (black), respectively. 449

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Aspergillus fumigatus (mode MIC 1 mg/l)

0 6 12 18 24 30 36 42 48 54 60 66 720

2

4

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Time (hours)

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Aspergillus flavus (mode MIC 1 mg/l)

0 6 12 18 24 30 36 42 48 54 60 66 720

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0 6 12 18 24 30 36 42 48 54 60 66 720

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12AMB Cmax=0.6 mg/lAMB Cmax=1.2 mg/lAMB Cmax=2.4 mg/lAMB Cmax=4.8 mg/l

Control w/o AMB

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Gal

acto

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450 451

Figure 2. Single-dose pharmacodynamic analysis of simulated amphotericin B doses with Cmax 0.6, 452

1.2, 2.4 and 4.8 mg/L against A. fumigatus, A. flavus and A. terreus isolates with mode CLSI MIC of 453

1 mg/l as determined by galactomannan index in the in vitro PK/PD model. 454

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In vitro pkd relationship

0

20

40

60

80

100A. fumigatusA. flavusA. terreus

4.82.41.20.60.30 9.6

A. fumigatusA. flavusA. terreus

Amphotericin B Cmax (mg/L)

% F

un

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(A

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456 457 458 Figure 3. Single-dose exposure-efficacy relationship of amphotericin B against each Aspergillus 459

species with modal CLSI MICs 1 mg/L for A. fumigatus, A. flavus and A. terreus in the in vitro 460

PK/PD system simulating amphotericin B human plasma levels based on the increasing 461

amphotericin B Cmaxs (maximum concentration) and the galactomannan index as a marker of fungal 462

growth. 463

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